The Biological Characterization Of The Vaccinia Virus Tian Tan (VTT) Strain And The Construction Of Attenuated VTT Vaccine Vectors By Genetic Engineering

Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that VTT has broad host cell range that 11 of the 12 mammalian cell lines studied and primary CEF are permissive to VTT infection whereas one, CHO-K1, is non-permissive. In addition, VTT produced severe cytopathic effects (CPE) in nearly all the tested cells, including 4 human cell lines. Moreover, VTT is much less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens.Vaccinia Hemagglutinin (HA) gene was reported highly associated with viral virulence in vivo and inactivation of HA gene resulted in varied extents of attenuation depending on the vaccinia strains. Here, we constructed recombinant TTzci and TT_pZCI by genetic modification in which HA gene was inactivated in TTzci genome while HA was partly inactivated in TT_pZCI by destroying its later promoter. The properties of the recombinants were systematically investigated regarding to the host cell range, replication in vitro, and virulence in vivo, and the data were compared with that of VTT carefully to reveal the property changes. We found HA modification resulted in varied decreases in viral replication in 7 of the 12 cell lines that are tested permissive for VTT replication, in which RK13 and MDCK became absolutely restricted for TTzci replication and RK13 failed in supporting TT_pZCI replication. Furthermore, diminished CPE and/or reduced cell-to-cell spread of the modified viruses occurred in most of the cells tested including human-derived cell lines compared with its parental VTT strain. In addition, the inserting and expressing stability of the inserted foreign gene (GFP) in rVTT's genomes were demonstrated through series passageing of the viruses in cultured cells. The virulence of the recombinant viruses was evaluated in animal model via different inoculation routes. We found that HA gene inactivation resulted in great attenuation of TTzci in mice model. In contrast, partly inactivation of HA gene let to obviously attenuation ofTT_pZCI although the attenuation extent was much less than that of TTzci- Further study revealed that the attenuated phenotype of TTzci was most probably attributed to the reduced replication of TTzci in nerve center in mice brain. The attenuated phenotype of TTzci will make it the attractive live viral vector for constructing safer vaccine candidates.Here, for the first time, we also demonstrated a previously unidentified function of vaccinia HA gene involving in host range determination in cell cultures. Different from previously reported orthopoxvirus host range genes, the HA gene product is required for vaccinia replication in both rabbit kidney cells and dog kidney cells. The underlying mechanisms of HA mutant virus in the host range restriction in cell cultures were further discussed.